Type I supersensitivity reactions are characteristic of atopic diseases. In terms of pathophysiology, the disease is attributed to disorders of immunological tolerance mechanisms, which have not been clarified in detail. The underlying immune reactions are mostly directed against harmless antigens that are tolerated by healthy subjects. These inflammation reactions are mediated by class E immunoglobulins. The IgE molecules are bound by mast cells and basophilic granulocytes through surface receptors. If, upon antigen contact, cross-linking between IgE/IgE receptor complexes occur, processes which lead to secretion of inflammation mediators are induced in the cells concerned. This is the starting point of the wide variety of symptoms observed in atopic diseases.
A humoral immune response begins with an IgM dominated primary antibody response. Later on, the antibody repertoire becomes specified by a switch recombination to the antibody isotypes IgG, IgA or IgE. These antibody classes are also employed for “memory antibodies” later. Having a higher affinity for a particular antigen is characteristic of the antibodies of the secondary immune response. The phenomenon on which this development is based is referred to as “affinity maturation of the antibody repertoire”. It is achieved by somatic mutations in the hypervariable regions of the variable regions of the heavy and light antibody chains and takes place within the germinal centers of lymphatic organs. The producers of the immunoglobulins are B lymphocytes whose maturation, differentiation and clonal expansion are essentially determined by the mutual contact with antigen-presenting cells and T helper (Th) cells. Thus, each B cell lineage expresses only one, genetically unique, variant of an immunoglobulin. Thus, the variability of different binding specificities is determined by the repertoire of different clonal origin cells. The mutual ratio of the sizes of the individual clones is determined by the dynamic adaptation to the current requirements. The specific binding activity towards antigens is established by the constitution of the variable regions of the light and heavy chains of an antibody.
Type 2 Th cells have a major influence on the development of IgE-secreting plasma cells. The orientation of immunological processes towards a particular antigen is ensured by highly specific surface receptors (B cell receptor, T cell receptor) on the B and Th lymphocytes, which enables the initial contact of both cell types only in the presence of an antigen for which both must have a specific receptor. This antigen-specific orientation of the immune reaction can be lost at high IgE titers when B cells of quite different specificities bind soluble immunoglobulins of these classes through the low-affinity receptor for IgE (CD23), and thus, in addition to their original specificity represented by the membrane-bound B cell receptor, recognize and internalize IgE-specific antigens and come into antigen-specific contact with Th2 cells through MHC II molecules. Thus, consequently, further IgE-secreting plasma cells are formed which can extend the hypersensitive antigen range extremely [1].
Although the participation of individual immunoglobulin classes in the atopic events has been described [2], the functional relationships between different classes of antibodies within the immune system, their influences on the overall structure, for example, within the scope of physiological and pathophysiological processes (such as atopic diseases, but also autoreactive reactions, sepsis, tolerance and anergy-controlling processes), are insufficiently understood. More profound insights into these processes can be derived from differentiated reaction profiles of the B cell lineages involved in the events, which may provide an image of the influence on the range of the antigen-specific V gene and antibody repertoires and isotype pattern in a functional relationship with the immunophysiological and pathophysiological status. To date, the enormous variability of the antibody repertoire has been a critical obstacle to the preparation of high-resolution representative reaction profiles.
Limited antigen-specific reaction profiles of antibodies are performed on the basis of serum analyses. Thus, the blood serum can be examined for reactivity against about 600 different allergens. However, this method does not provide any information about non-secreting B cells which are inhibited from secreting antibodies by suppressive influences. Thus, not all potentially reactive cells are covered, and it is not possible with this method to determine the presence of a predisposition for immune diseases.
Further, it is possible to determine the antigen-specific reaction profile directly at the patient, in vivo, by skin prick, intracutaneous and challenge tests. These methods have drawbacks in that they put a load on the patient and can be applied only to a limited extent with highly allergic subjects.
Antigen-specific V gene profiles based on single cell analyses have also been described [3, 4]. These approaches are suitable for a representative portrayal of the antigen-specific V gene repertoire, but have not been used for a comprehensive isotype-specific analysis of the V gene repertoires in this form.
Recently, also in the mouse model system, B cells which are undergoing the affinity maturation process (somatic hypermutation) could be successfully isolated. A protocol was developed which enabled the subsequent isolation of B cells by means of FACS. From the hapten- or allergen-specific cells, RNA was isolated and subsequently subjected to reverse transcription into cDNA. Subsequently, allergen/hapten-specific heavy chains of immunoglobulins could be isolated from the cDNA by means of RT-PCR. The sequences were then subjected to an intensive computer analysis. The following results were achieved:                Without any doubt, the IgE response is able to “mature”.        The somatic diversity is different from isotype to isotype.        The antigen receptor is necessary for the selection of high affinity antibodies.        The antigen receptor has no function for the regulation of the somatic mutation process.        The shorter the CDR3 region, the higher the affinity of the antibodies for the hapten.        IgE does not form any “long lived” plasma cells.        The IgE response has a short life.        The cytoplasmic “tail” seems to be a suitable target for allergy therapy.        
The results of this partial project have been published in the “European Journal of Immunology” [5].
Thus, due to the enormous variability of the antibody repertoire, it has been problematic or impossible to determine a comprehensive antigen-specific reaction profile of an organism. In particular, there has been no low risk method that puts a low load on the patient for evaluating their allergic (antigen-specific) reaction profile. Surprisingly, the present invention offers a solution to this problem. The subject matter of the invention of the present application allows the antigen-specific reaction profile of an organism to be determined on a genetic level. Surprisingly, it is possible in this way to cover all B-cell-mediated antigen-specific reactions, especially including those in which the secretion activity of the B cells is inhibited by suppressive influences.